The absence of genotoxicity of a mixture of aloin A and B and a commercial aloe gel beverage

Abstract

Aloe products are increasingly valued as ingredients in food supplements and as flavoring agents. The global Aloe vera market is varied, large, growing, and increasingly important in food, cosmetics, and medicines. Aloin, an anthraquinone glycoside, is one of the major components by weight of the anthraquinone derivatives of Aloe vera gel. Principal metabolites, aloe emodin and emodin, are a source of debate concerning toxic vs salutary effects, hence the accurate toxicological characterization of these compounds has become increasingly important. The purpose of this study was to determine the genotoxic profile of a stabilized Aloe vera juice product derived from the inner filet and marketed as a beverage currently sold in the European Union containing 8 to 10 ppm aloin and a mixture of purified aloin A and B. The present data confirm that a commercial stabilized Aloe vera gel intended for consumption as a juice beverage is not genotoxic. Furthermore, both aloin A and B were negative in the same assays and therefore are also not genotoxic. These results are consistent with the work of other groups and contrast with data obtained using products containing the Aloe vera latex hydroxyanthracene derivatives (HADs).

Keywords:

• Ames
• microcucleus
• aloin
• aloe
• hydroxyanthracene

Introduction

Aloe vera is one of more than 400 species of the genus Aloe. Aloe vera specifically refers to the Aloe barbadensis Miller plant, which is the most common species used in Aloe-based products. The global Aloe vera market was valued at between USD 600 million and 1.60 billion in 2018 and is anticipated to expand at a compound annual growth rate of between 7.6% and 8.5% (https://www.grandviewresearch.com/industry-analysis/aloe-vera-extracts-market). Growing awareness regarding the putative medicinal importance of Aloe vera in the management of various pathologies has increased the demand for Aloe vera extracts in natural and organic cosmetics, dietary supplements, beverages, and other consumer products (Pressman et al. 2019). One of the major trends seen in the global market is the increasing use of Aloe vera extracts in sugar-free drinks. Development in this space is expected to contribute to new formulations and novel products in the global food markets. However, the range of aloe-containing products is not alike, and there is significant diversity in terms of the chemical composition, especially regarding the hydroxyanthracene derivative (HAD) content, depending on the portion of the plant used, the method of extraction, preparation, and processing used in the manufacturing of these products. Salient for the present study is the fact that the inner leaf of the Aloe vera plant consists of aqueous parenchyma, from which the gel may be collected. In addition to water, the Aloe vera gel contains various polysaccharides, notably acemannan. This compound has been associated with an array of health-promoting properties supported by both in vitro and in vivo studies (Im et al. 2010; Kumar and Tiku 2016; Quezada et al. 2017).

In the last few years, there has been much research on aloe in commerce, prompted not only by its popularity but by a recent opinion of the European Food Safety Authority (EFSA) in 2017 (https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2018.5090) in which the Food Additives and Nutrient Sources Added to Food (ANS) Panel of the European Food Safety Authority (EFSA) declared that hydroxyanthracene derivatives should be “considered as genotoxic and carcinogenic unless there are specific data to the contrary, … and that there is a safety concern for extracts containing hydroxyanthracene derivatives although uncertainty persists” (Younes et al. 2021). The European Commission subsequently asked EFSA to assess the safety of these plant ingredients when used in foods, and provide advice on a daily intake not associated with adverse health effects. Based on the data available at that time, EFSA concluded that certain hydroxyanthracene derivatives were genotoxic (Mortensen et al. 2018). Therefore, it was not possible to set a safe daily intake.

Moreover, the European Commission decided to place aloe-emodin and all the extracts in which this substance is present in Part A (ban on the use in food) of Annex III of Regulation (EC) No. 1925/2006 of the European Parliament and the Council to ensure a high level of health protection per the precautionary principle provided for in Article 7 of Regulation (EC) 178/2002 (Commission Regulation (EU) 2021/468 of March 18, 2021).

Baldi et al (2021) provided a comprehensive review of the toxicological aspects of aloe gel-based products and briefly discuss the chemical profile of other Aloe vera preparations. Aloe-based products represent a large and growing market, within which there is great variety in the composition and character of the Aloe vera. Unlike many Aloe vera products, pure Aloe vera gel shows no toxic effects. However, and again in light of the EFSA opinion, these investigators called for a further toxicological evaluation of specific commercial products to more accurately establish the maximum permissible limit of HADs in the Aloe vera gel. We concur that rigorous market surveillance in the form of careful monitoring of the HAD content in consumable Aloe products is necessary to assure public health.

Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion was based, Galli et al. (2021) conducted an in vivo study (in vivo alkaline comet assay in mice—OECD 489) to test the potential genotoxicity of aloe-emodin (97.12% purity) on preparations of single cells from the kidney and colon of male mice treated orally with doses of 0, 250, 500, 1000 and 2000 mg/kg bw/day. Following treatment with the test item, no significant clinical signs were observed in any of the animals in any treatment group. Under these experimental conditions, aloe-emodin showed no genotoxic activity.

Aloin (C₂₁H₂₂O₉) is a yellow aromatic organic compound, composed of two diastereoisomers, aloin A and aloin B. Aloin is a phytoconstituent of aloes, an anthrone C-glucoside with a molecular weight of 418. It is a constituent of a larger family of compounds known as hydroxyanthracene derivatives (HADs). EFSA concluded, from their analysis of the literature, that Aloe extracts were genotoxic in vitro. This observation was attributed to the presence of hydroxyanthracene derivatives (HADs). In particular, the aloin concentration is putatively considered the dominant factor in the toxicological profile. This point is supported by the literature, which reports the genotoxic and carcinogenic potential of aloin, its metabolites, and structural analogs (Mori et al. 1985, 1986; Heidemann et al. 1993; Mueller et al. 1999; Nesslany et al. 2007; Chen et al. 2010; Boudreau et al. 2017).

The impact of the intestinal flora is a central part of the story. Boudreau et al. (2017) point out that due to the C-glycosidic bond, and the anthrone ring structure, as well as the hydrophilic nature of the compound, aloin is protected from acid hydrolysis in the stomach, and reaches the large intestine in an undigested form where bacteria metabolize it to yield glucose and aloe-emodin-9-anthrone which is then oxidized to aloe-emodin (with its putative mutagenic and cell transforming properties). Therefore, the reduction of aloins A and B appears to be a priority (Huang et al. 2019).

To confirm the Galli et al. (2021) negative in vivo alkaline comet assay in mice and to further refute the marked uncertainty reported in the EFSA ANS Panel opinion, we conducted a battery of in vitro genotoxic assays with both a commercial aloe gel beverage currently sold in the European Union containing 8 to 10 ppm aloin and a mixture of purified aloin A and B. The battery included an Ames Assay, an in vitro cytotoxic assay, and an in vitro mammalian cell micronucleus assay in human peripheral blood lymphocytes.

Materials and methods

Materials

All assays were conducted at BioReliance, Rockville, MD, USA following Organization for Economic Cooperation and Development testing guidelines and US FDA Good Laboratory Practice for Non-Clinical Laboratory Studies as published in 21 CFR 58.

The Aloe vera inner leaf filet gel juice in the form of a commercial-ready stable Aloe vera gel beverage containing 8 to 10 ppm aloin was obtained from Forever Living, Scottsdale, Arizona. A single sample was used for all the assays. Aloe vera latex naturally contains anthraquinones but the Aloe vera gel does not. However, the gel may be contaminated with anthraquinones from the latex if not properly processed. The Forever Living production process is a thermal phase or "stabilizing" process in which the Aloe vera gel [innermost pulp of the leaf; not the latex or juice] is subject to rapid heating from 35 to 80 °C (US Patent, Maughan et al 6,713,095, March 30, 2004). Thermal effects at 80 °C inactivate intrinsic amylase [degrade at >40 degrees C (Hwang et al. 2013)]. The aloe juice beverage tested was certified by the International Aloe Scientific Council (IASC) to meet the quality standards for microbiology testing, production, storage, Aloe vera content, and aloin content.

Aloin A (95% purity) was certified by the United States Pharmacopeia (USP, Rockville, MD, USA) while Aloin B (91% purity) was certified by ChromaDex (Irvine, CA, USA). All other chemicals and materials were of the highest available purity and met the standards for GLP and OECD testing guidelines. Sterile water was the vehicle. Both negative and positive controls were used in all test systems as appropriate. The Ames Assay, the cytotoxic assay, and the in vitro mammalian cell micronucleus assay were conducted by exposing selected strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and the tryptophan locus of Escherichia coli strain WP2 uvrA, mouse fibroblasts (BALB/c 3T3 clone A31; CCL-163), and human peripheral blood lymphocytes (HPBL), respectively, to appropriate concentrations of the test articles as well as the concurrent positive, negative and/or vehicle controls, in the presence and absence of an exogenous rat metabolic activation system.

Assays

The battery of assays conducted included: Ames Assay (OECD TG 471), in vitro cytotoxic assay (ICCVAM, 2006), and in vitro mammalian cell micronucleus assay in human peripheral blood lymphocytes (OECD TG 587).

Genotoxicity testing of the beverage

The top dose tested in all three assays was the undiluted, neat commercial Aloe vera beverage as currently sold in the European Union with an aloin content between 8 and 10 ppm. Each assay met all the criteria necessary for a valid assay.

Neutral Red Uptake (NRU) in vitro cytotoxicity assay: The undiluted/neat beverage was the top dose tested using BALB/c 3T3 cells in the Neutral Red Uptake (NRU) in vitro cytotoxicity assay (NIH Publication No. 07-4519, ICCVAM 2006). The NRU test is a standard test for the cytotoxicity evaluation of chemicals (Liebsch and Spielmann 1995; Thome et al. 2014).

Mouse fibroblasts (BALB/c 3T3 clone A31; CCL-163) (American Type Culture Collection [ATCC], Manassas, VA, USA) were maintained in Dulbecco's Modified Eagle Medium (DMEM; containing 4 mM glutamine and 4.5 g/L glucose supplemented with 10% heat-inactivated newborn calf serum (HI-NCS) at 37 ± 1 °C in an atmosphere of 5% CO₂. Exponentially growing BALB/c 3T3 cells were diluted in 3.0 × 10⁴ cells/mL in the tissue culture medium on the day of plate seeding. Using a multichannel pipette, 100 µL of tissue culture medium was dispensed into the peripheral wells (blanks) of a 96-well tissue culture microtiter plate. Then, 100 µL of a cell suspension of 2.0–3.0 × 10⁴ cells/mL (2.0–3.0 × 10³ cells/well) was dispensed in the remaining wells. The cultures were incubated under standard conditions for 24 ± 2 hours. Following treatment, the medium was carefully decanted, and cells were washed with 150 µL prewarmed Calcium–Magnesium Free Phosphate-Buffered Saline (CMF-PBS). Two hundred fifty microliters (250 µL) neutral red (NR) medium (25 µg NR/mL, containing 5% HI-NCS, penicillin/streptomycin/L-glutamine) was added to each well and incubated at 37 °C in a humidified atmosphere under standard culture conditions for 3 ± 0.1 hours. After incubation, the NR medium was removed. Cells were washed with 150 µL pre-warmed CMF-PBS. The culture plate was inverted to remove the CMF-PBS. A one hundred microliter (100 µL) NR Desorb (EtOH/Water/Acetic acid: 50%/49%/1¾) solution was added to all wells, including blanks. The light-protected plate (foil wrapped) was shaken rapidly (1050 rpm) on a microtiter plate shaker until the NR extracted from the cells formed a homogeneous solution. Plates were allowed to stand for at least 5 minutes and any bubbles were ruptured before measuring optical density. Within 60 minutes of adding the Neutral Red Desorb Solution, the optical density (OD) of the cells in each well was measured at 540 nm ± 10 nm in a microtiter plate reader. The data from six replicate samples were analyzed for the concentration-response and calculation of IC₅₀/EC₅₀. The average of the six replicates of the test article and the vehicle control was calculated and plotted for dose-response manually. Based upon the quantitative cytotoxicity results, the effective concentration that led to 50% cell death (IC50/EC50) was determined.

Ames assay

The neat/undiluted beverage was evaluated for its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of a rat exogenous metabolic activation system following OECE TG 471. A minimum of three nontoxic dose levels is required to evaluate the assay data. For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test article as specified. Strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 3.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range. Strains TA98, TAlO0, and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 2.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range.

In vitro mammalian cell micronucleus assay

The undiluted/neat Aloe vera Gel beverage was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous rat metabolic activation system (OECD TG 587). HPBL were treated for four hours in the absence and presence of S9, and for 24 hours in the absence of S9. Cytotoxicity [55 ± 5% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose in any of the treatment groups. At least 2000 binucleated cells from at least three appropriate test article concentrations were evaluated. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria: the micronucleus should have the same staining characteristics as the main nucleus, the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges), and the micronuclei should be of regular shape and approximately one-third or less than the diameter of the main nucleus. The test article was considered to have induced a positive response if at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), the increase was concentration-related (p ≤ 0.05), and the results were outside the 95% control limit of the historical negative control data. The test article was considered to have induced a clear negative response if none of the criteria for a positive response was met.

Genotoxicity testing of the aloin mixture

Dose formulations were prepared on the day of use. The top aloin concentration tested was a mixture of an equal amount of aloin A and aloin B, at a 1:1 ratio by weight, dissolved in the vehicle, sterile water. All subsequent dosing concentrations were prepared by serial dilution. Test article dilutions were prepared immediately before use and delivered to the test system at room temperature (23-25 °C) under filtered light.

Ames assay

The test article, a one-to-one mixture of Aloin A and B, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of a rat exogenous metabolic activation system. Sterile water was the vehicle. The dose levels tested were 15.0, 50.0, 150, 500, 1500, and 5000 µg (limit dose) per plate. Neither precipitate nor toxicity was observed. The study met all the criteria for a valid assay.

In vitro mammalian cell micronucleus assay

The doses selected for evaluation of micronuclei were 0, 198,354, and 486 µg/mL for the non-activated four-hour exposure group; 0, 198, 354, and 437 µg/mL for the nonactivated 24-hour exposure group; and 0, 220, 550, and 750 µg/mL for the S9-activated four-hour exposure group. Slides from at least three test article treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) was examined and scored for the presence of micronuclei by the same individual. The test article was considered to have induced a clear negative response if none of the criteria for a positive response was met (see criteria for a positive response for this assay under genotoxicity testing of the beverage). The study met all the criteria for a valid assay.

Data analysis

Statistical analysis for the in vitro mammalian cell micronucleus assay was performed using Fisher's exact test (p: S 0.05) for pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran–Armitage trend test was used to assess dose-responsiveness. For the cytotoxicity assay, the average of the six replicates of test article and vehicle control was calculated and plotted for dose–response manually using an Excel file. Based upon the quantitative cytotoxicity results, the effective concentration that led to 50% cell death was determined (i.e., the IC₅₀/EC₅₀).

Results

Beverage

Cytotoxicity in BALB/c 3T3 cells using neutral red dye uptake

The viability of cells exposed to the test article was greater than 50% at all concentrations tested (data not shown); thus, an IC₅₀ was not calculated. All criteria for a valid test were met.

Ames assay

The results of the mutagenicity assay are presented in Tables 1 and 2. Neither precipitate nor toxicity was observed. o mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. All criteria for a valid test were met.

Table 1. Mutagenicity assay without S9 activation.

Strain	Article	Dose Level per Plate	Mean Revertants per Plate	Standard Deviation	Ratio Treated/Solvent	Individual Revertant Colony Counts and Background Codes^a
TA 98	Aloe Vera Gel Beverage	1000 μL	19	7	1.4	16^A, 27^A, 14^A
		750 μL	11	3	0.8	10^A, 9^A, 15^A
		500 μL	16	4	1.1	19^A, 17^A, 11^A
		250 μL	12	1	0.9	11^A, 13^A, 13^A
		100 μL	11	2	0.8	10^A, 10^A, 14^A
	Water	50 μL	14	1	1.0	13^A, 14^A, 14^A
		1000 μL	14	4		14^A, 10^A, 17^A
TA100	Aloe Vera Gel Beverage	1000 μL	126	5	1.3	129^A, 120^A, 128^A
		750 μL	107	19	1.1	107^A, 88^A, 125^A
		500 μL	110	16	1.2	128^A, 105^A, 97^A
		250 μL	101	7	1.1	106^A, 104^A, 93^A
		100 μL	97	10	1.0	86^A, 101^A, 104^A
	Water	50 μL	96	13	1.0	97^A, 83^A, 109^A
		1000 μL	93	14		111^A, 84^A, 90^A
TA1535	Aloe Vera Gel Beverage	1000 μL	12	2	1.3	13^A, 10^A, 14^A
		750 μL	10	1	1.1	9^A, 10^A, 11^A
		500 μL	12	5	1.3	7^A, 11^A, 17^A
		250 μL	13	4	1.4	15^A, 9^A, 16^A
		100 μL	11	4	1.2	15^A, 10^A, 7^A
	Water	50 μL	8	2	0.9	9^A, 5^A, 9^A
		1000 μL	9	2		11^A, 8^A, 7^A
TA1537	Aloe Vera Gel Beverage	1000 μL	7	4	1.0	6^A, 3^A, 11^A
		750 μL	10	6	1.4	15^A. 13^A, 3^A
		500 μL	8	3	1.1	7^A, 11^A, 5^A
		250 μL	6	1	0.9	6^A, 5^A, 7^A
		100 μL	8	4	1.1	6^A, 6^A, 13^A
	Water	50 μL	10	4	1.4	5^A, 11^A, 13^A
		1000 μL	7	3		7^A, 10^A, 5^A
WP2uvrA	Aloe Vera Gel Beverage	1000 μL	42	6	1.4	46^A, 35^A, 44^A
		750 μL	27	3	0.9	29^A. 29^A, 24^A
		500 μL	27	8	0.9	23^A, 36^A, 23^A
		250 μL	25	2	0.8	27^A, 23^A, 25^A
		100 μL	21	5	0.7	25^A, 23^A, 15^A
	Water	50 μL	23	5	0.7	29^A, 19^A, 21^A
		1000 μL	31	8		25^A, 40^A, 27^A
TA98	2NF	1.0 μg	138	6	9.9	138^A, 144^A, 133^A
TA100	SA	1.0 μg	772	61	8.1	759^A. 838^A, 719^A
TA1535	SA	1.0 μg	687	10	76.3	678^A, 697^A, 687^A
TA1537	9AAD	75.0 μg	898	19	128.3	913^A, 905^A, 876^A
WP2uvrA	MMS	1000 μg	437	40	14.1	402^A, 428^A, 481^A
Key to Positive Controls
2NF	2 nitrofluorene	9AAD	9-aminoacridine
SA	sodium azide	MMS	methyl methane sulfonate

^aA: Automatic Count.

Table 2. Mutagenicity assay with S9 activation.

Strain	Article	Dose Level per Plate	Mean Revertants per Plate	Standard Deviation	Ratio Treated / Solvent	Individual Revertant Colony Counts and Background Codes^a
TA 98	Aloe Vera Gel Beverage	1000 μL	32	5	1.4	29^A, 30^A, 38^A
		750 μL	25	12	1.1	27^A, 13^A, 36^A
		500 μL	32	8	1.4	25^A, 41^A, 31^A
		250 μL	27	5	1.2	24^A, 32^A, 24^A
		100 μL	22	10	1.0	26^A, 29^A, 11^A
	Water	50 μL	20	3	0.9	17^A, 22^A, 22^A
		1000 μL	23	6		17^A, 27^A, 26^A
TA100	Aloe Vera Gel Beverage	1000 μL	131	21	1.1	143^A, 143^A, 107^A
		750 μL	124	7	1.1	116^A, 129^A, 128^A
		500 μL	118	15	1.0	133^A, 116^A, 104^A
		250 μL	139	9	1.2	138^A, 149^A, 131^A
		100 μL	129	7	1.1	122^A, 135^A, 131^A
	Water	50 μL	132	5	1.1	129^A, 129^A, 137^A
		1000 μL	118	13		106^A, 131^A, 137^A
TA1535	Aloe Vera Gel Beverage	1000 μL	13	2	1.1	14^A, 13^A, 11^A
		750 μL	13	3	1.1	9^A, 14^A, 15^A
		500 μL	11	3	0.9	10^A, 8^A, 14^A
		250 μL	16	9	1.3	13^A, 9^A, 26^A
		100 μL	9	2	0.8	10^A, 10^A, 7^A
	Water	50 μL	10	3	0.8	10^A, 13^A, 8^A
		1000 μL	12	4		17^A, 11^A, 9^A
TA1537	Aloe Vera Gel Beverage	1000 μL	10	5	1.3	13^A, 13^A, 5^A
		750 μL	11	4	1.4	10^A, 8^A, 16^A
		500 μL	13	4	1.6	16^A, 9^A, 15^A
		250 μL	9	2	1.1	8^A, 11^A, 9^A
		100 μL	14	3	1.8	11^A, 16^A, 14^A
	Water	50 μL	9	3	1.1	13^A, 11^A, 6^A
		1000 μL	8	2		8^A, 6^A, 9^A
WP2uvrA	Aloe Vera Gel Beverage	1000 μL	39	1	1.1	38^A, 40^A, 40^A
		750 μL	31	1	0.9	30^A, 32^A, 32^A
		500 μL	32	6	0.9	31^A, 39^A, 27^A
		250 μL	42	6	1.2	41^A, 36^A, 48^A
		100 μL	30	5	0.9	25^A, 34^A, 32^A
	Water	50 μL	33	9	0.9	23^A, 35^A, 41^A
		1000 μL	35	3		33^A, 34^A, 38^A
TA98	2AA	2.0 μg	833	54	36.2	844^A, 881^A, 774^A
TA100	2AA	5.0 μg	2610	142	22.1	2771^A, 2554^A, 2505^A
TA1535	2AA	2.0 μg	155	37	12.9	197^A, 140^A, 127^A
TA1537	2AA	5.0 μg	193	17	24.1	190^A, 211^A, 177^A
WP2uvrA	2AA	15.0 μg	320	39	9.1	296^A, 365^A, 298^A
Key to Positive Controls
2AA	2-aminoanthracene

^aA: Automatic Count.

In vitro mammalian cell micronucleus assay

Cytotoxicity (55 ± 5% CBPI relative to the vehicle control) was not observed at any dose in any of the treatment groups. Micronucleus data for individual exposure groups are presented as follows: non-activated 4-hour exposure in Table 3; S9-activated four-hour exposure in Table 4; non-activated 24-hour exposure in Table 5. Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher's Exact and Cochran–Armitage tests). The results were within the 95% control limit of the historical negative control data. All criteria for a valid test were met.

Table 3. Micronucleus analysis of human peripheral blood lymphocytes treated with Aloe Vera gel beverage (FLP-AVG) in the absence of exogenous metabolic activation: 4-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.10%	0.15%
	B	1000	0.20%
Aloe Vera Gel Beverage (FLP-AVG)
0.25 p	A	1000	0.20%	0.15%
	B	1000	0.10%
0.5 p	A	1000	0.10%	0.15%
	B	1000	0.20%
0.947 (Neat) p	A	1000	0.10%	0.15%
	B	1000	0.20%
MMC, 0.5 μg/mL	A	1000	2.10%	2.30%^a
	B	1000	2.50%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

Table 4. Micronucleus analysis of human peripheral blood lymphocytes treated with Aloe Vera gel beverage (FLP-AVG) in the presence of exogenous metabolic activation: 4-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.20%	0.20%
	B	1000	0.20%
Aloe Vera Gel Beverage (FLP-AVG)
0.25 p	A	1000	0.20%	0.20%
	B	1000	0.20%
0.5 p	A	1000	0.10%	0.10%
	B	1000	0.10%
0.947 (Neat) p	A	1000	0.10%	0.15%
	B	1000	0.20%
CP, 7.5 μg/mL	A	1000	1.40%	1.35%^a
	B	1000	1.30%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

CP: Cyclophosphamide.

Table 5. Micronucleus analysis of human peripheral blood lymphocytes treated with Aloe Vera gel beverage (FLP-AVG) in the absence of exogenous metabolic activation: 24-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.10%	0.15%
	B	1000	0.20%
Aloe Vera Gel Beverage (FLP-AVG)
0.25 p	A	1000	0.20%	0.20%
	B	1000	0.10%
0.5 p	A	1000	0.20%	0.10%
	B	1000	0.20%
0.947 (Neat) p	A	1000	0.20%	0.20%
	B	1000	0.20%
CP, 7.5 μg/mL	A	1000	2.00%	1.90%^a
	B	1000	1.80%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

CP: Cyclophosphamide.

Aloin mixture

Ames assay

The results of the mutagenicity assay are presented in Tables 6 and 7. Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500, and 5000 µg per plate. No precipitate was observed. Toxicity as a reduction in the revertant count was observed at 2: 1500 and 5000 µg per plate with tester strains TA98 and TAl00 in the presence of S9 activation, respectively. No mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The results for the controls indicate that all criteria for a valid assay were met.

Table 6. Mutagenicity assay without S9 activation.

Strain	Article	Dose Level per Plate	Mean Revertants per Plate	Standard Deviation	Ratio Treated / Solvent	Individual Revertant Colony Counts and Background Codes^a
TA 98	Aloin A and B	5000 μL	16	1	0.8	17^A, 15^A, 16^A
		1500 μL	16	6	0.8	20^A, 10^A, 19^A
		500 μL	15	3	0.8	15^A, 12^A, 17^A
		150 μL	17	5	0.9	20^A, 20^A, 12^A
		50 μL	14	2	0.7	12^A, 14^A, 15^A
		15 μL	14	3	0.7	17^A, 11^A, 15^A
	Water	200 μL	20	5		14^A, 21^A, 24^A
TA100	Aloin A and B	5000 μL	105	17	1.1	123^A, 103^A, 90^A
		1500 μL	110	13	1.1	124^A, 108^A, 99^A
		500 μL	103	4	1.0	100^A, 102^A, 108^A
		150 μL	114	4	1.1	119^A, 113^A, 111^A
		50 μL	113	7	1.1	116^A, 118^A, 105^A
		15 μL	90	14	0.9	106^A, 79^A, 85^A
	Water	200 μL	100	19		85^A, 121^A, 94^A
TA1535	Aloin A and B	5000 μL	15	6	1.9	14^A, 21^A, 9^A
		1500 μL	12	6	1.5	16^A, 5^A, 15^A
		500 μL	12	5	1.9	19^A, 16^A, 9^A
		150 μL	12	2	1.5	12^A, 14^A, 11^A
		50 μL	12	3	1.5	12^A, 9^A, 14^A
		15 μL	12	3	1.5	12^A, 15^A, 9^A
	Water	200 μL	8	3		7^A, 12^A, 6^A
TA1537	Aloin A and B	5000 μL	7	2	0.9	9^A, 6^A, 6^A
		1500 μL	8	4	1.0	4^A, 12^A, 9^A
		500 μL	7	2	0.9	5^A, 6^A, 9^A
		150 μL	5	3	0.6	7^A, 7^A, 2^A
		50 μL	7	2	0.9	9^A, 6^A, 7^A
		15 μL	7	3	0.9	6^A, 5^A, 11^A
	Water	200 μL	8	2		7^A, 10^A, 7^A
WP2uvrA	Aloin A and B	5000 μL	32	5	0.7	36^A, 33^A, 26^A
		1500 μL	33	6	0.8	32^A, 40^A, 28^A
		500 μL	41	4	0.9	43^A, 36^A, 43^A
		150 μL	38	6	0.9	41^A, 31^A, 41^A
		50 μL	43	2	1.0	42^A, 45^A, 42^A
		15 μL	35	6	0.8	31^A, 42^A, 31^A
	Water	200 μL	44	12		33^A, 56^A, 43^A

^aA: Automatic Count.

Table 7. Mutagenicity assay with S9 activation.

Strain	Article	Dose Level per Plate	Mean Revertants per Plate	Standard Deviation	Ratio Treated / Solvent	Individual Revertant Colony Counts and Background Codes^a
TA98	2AA	2.0 μg	597	26	21.3	598^A, 623^A, 571^A
TA100	2AA	5.0 μg	2740	37	28.5	2776^A, 2741^A, 2703^A
TA1535	2AA	2.0 μg	106	9	8.8	116^A, 103^A, 98^A
TA1537	2AA	5.0 μg	194	10	16.2	201^A, 198^A, 183^A
WP2uvrA	2AA	15.0 μg	273	20	5.6	250^A, 286^A, 284^A
Key to Positive Controls
2AA	2-aminoanthracene

^aA: Automatic Count.

In vitro mammalian cell micronucleus assay

The test article was soluble in the treatment medium at all doses tested. The pH of the highest dose of the test article in the treatment medium was 7.5. Cytotoxicity (2: 55 ± 5% reduction in CBPI relative to the vehicle control) was observed at doses 2: 700 µg/mL in the S9-activated 4-hour exposure group. Micronucleus data for individual exposure groups are presented as follows: non-activated 4-hour exposure in Table 8; S9-activated four-hour exposure in Table 9; nonactivated 24-hour exposure in Table 10. Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in the treatment groups with or without S9 (p > 0.05). The results were within the 95% control limit of the historical negative control data. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Table 8. Micronucleus analysis of human peripheral blood lymphocytes treated with a one to one mixture of aloin A and B in the absence of exogenous metabolic activation B1 assay: 4-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.30%	0.35%
	B	1000	0.40%
Aloin A and B
198	A	1000	0.30%	0.35%
	B	1000	0.40%
354	A	1000	0.30%	0.30%
	B	1000	0.30%
486	A	1000	0.50%	0.45%
	B	1000	0.40%
MMC, 0.5 μg/mL	A	1000	1.90%	1.75%^a
	B	1000	1.60%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

MMC: Mitomycin C.

Table 9. Micronucleus analysis of human peripheral blood lymphocytes treated with a one to one mixture of aloin A and B in the presence of exogenous metabolic activation B2 assay: 4-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.30%	0.30%
	B	1000	0.30%
Aloin A and B
220	A	1000	0.50%	0.50%
	B	1000	0.50%
550	A	1000	0.30%	0.25%
	B	1000	0.30%
750	A	1000	0.40%	0.45%
	B	1000	0.50%
CP, 7.5 μg/mL	A	1000	1.30%	1.20%^a
	B	1000	1.10%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

CP: Cyclophosphamide.

Table 10. Micronucleus analysis of human peripheral blood lymphocytes treated with a one to one mixture of aloin A and B in the absence of exogenous metabolic activation B1 assay: 24-hour treatment, 24-hour harvest.

Treatment (ppm)	Replicate Culture	Total Number of Cells Counted	Percentage of Micronucleated Binucleated Cells per Culture	Average Percent Micronucleated Binucleated Cells per Dose
Sterile Water	A	1000	0.50%	0.45%
	B	1000	0.40%
Aloin A and B
198	A	1000	0.20%	0.25%
	B	1000	0.30%
354	A	1000	0.40%	0.35%
	B	1000	0.30%
437	A	1000	0.30%	0.30%
	B	1000	0.30%
VB, 10 ng/mL	A	1000	2.30%	2.10%^a
	B	1000	1.90%

^ap ≤ 0.01, Fisher’s Exact test, relative to solvent control.

VB: Vinblastine.

Discussion

Although Boudreau et al. (Boudreau et al. 2017) suggested that aloin, a component of the Aloe Vera plant leaf, can induce pathological changes and modulate the composition of the microbiota in the large intestines of F344/N male rats, Baldi and his colleagues carefully reviewed the literature and concluded that Aloe vera gel does not present toxic effects, unlike whole leaf extracts or Aloe latex (Baldi et al. 2021).

Since the EFSA ANS Panel opined that all hydroxyanthracene derivatives should be considered as genotoxic and carcinogens “unless there are specific data on the contrary” (Younes et al. 2021), and in light of the epidemiologic and growing toxicologic data to the contrary, it seemed imperative to rigorously surveil and test products that have been publicly embraced. One of the gaps for the safety profile was a battery of genotoxicity assays of the widely popular Aloe vera Gel beverage sold in the European Union and the two primary constituent HADs, Aloin A and B. This work has been completed and reported herein. In all cases, the results were conclusive, negative, and supportive of the safety of the Aloe vera gel beverage.

The battery of tests included the Ames test, an in vitro micronucleus assay with human peripheral blood lymphocytes, and a cytotoxicity test and was conducted following OECD guidelines in a GLP-certified laboratory. These tests verified that the Aloe vera Gel beverage containing 8–10 ppm of Aloin A and B plus other HADs at less than 0.5 ppm was negative in tests for damage to the nucleic acid, DNA, and the chromosomes. The mixture of purified Aloin A and B was also negative. These results confirm that neither the Aloe vera Gel beverage nor the Aloin mixture was genotoxic because neither induced mutation or damaged chromosomes in these assays.

Although there are, at best, only limited epidemiological data available regarding the toxicity and/or carcinogenicity of Aloe vera (Aloe barbadensis Miller), adverse effects in rodents have raised questions of its potential toxicity in humans (Guo and Mei 2016). To confound the issue, most toxicity and safety assessment studies in animals have been conducted on the non-decolorized whole leaf extract (WLE) which contains several latex compounds, such as phenolics that include anthraquinone C-glycosides, anthrones, and free anthraquinones (Boudreau et al. 2013). Furthermore, most of the studies dealing with the toxicity and safety of Aloe vera were difficult to interpret because the specific aloe extract was not identified or fully characterized, or the content and/or concentration of the hydroxyanthracene glycosides, especially emodin or aloin A (barbaloin), were not reported.

Some studies have suggested that Aloe vera exposure causes toxicity while other studies have reported no untoward effects. For example, the hydroxyanthracene glycosides in Aloe vera have been reported to be genotoxic to bacteria and mammalian cells (Boudreau et al. 2013). A 2013 report, however, indicated that decolorized Aloe vera juice did not increase mutagenesis using the Salmonella typhimurium TAl00 strain (Sehgal et al. 2013a). A DNA damage repair assay demonstrated the absence of a dose-related increase in SOS transgene induction. In addition, the juice did not induce DNA damage repair in the presence of S9 extract; however, at high concentrations, the juice appeared to be either cytotoxic or bacteriostatic in the Escherichia coli system. The investigators concluded that the effect on bacterial growth was not indicative of increased DNA damage.

In a frequently cited toxicology study of commercial decolorized Aloe vera juice, whole-leaf Aloe vera was treated with activated charcoal to remove the latex portion of the plant and then evaluated for genotoxicity and mammalian toxicity (Sehgal et al. 2013a). The processed juice was non-genotoxic both in histidine reversion (Ames Assay) and DNA repair assays. When fed to male and female F344 rats over 13 weeks, decolorized whole leaf aloe produced no toxicity as assessed by behavior, stools, weight gain, feed consumption, organ weights, and hematologic or clinical chemistry profiles (Sehgal et al. 2013a). Sehgal and colleagues (Sehgal et al. 2013b) also evaluated a stabilized Aloe vera gel supplement drink in mice. The supplement drink was negative in Salmonella TA 100 and did not increase the SOS DNA repair response in Escherichia coli or toxicity in B6C3F1 mice.These results were opposed to those obtained using preparations containing aloe latex phenolic compounds such as anthraquinones reported in the study conducted by the National Toxicology Program (Boudreau et al. 2013).

Another approach to assessing the safety of Aloe vera gel involved supercritical CO₂ extraction (Tanaka et al. 2012). This gel was negative in the bacterial reverse mutation test using S. typhimurium strains (TA98, TAl00, TA1535, and TA 1537) and in E.coli (Wp2uvrA) with and without metabolic activation up to 5000 µg/plate. The extract was also negative in a chromosomal aberration test in Chinese hamster lung cells at 1600 µg/plate and an in vivo bone marrow micronucleus test at 150 mg/kg/day. The supercritical CO₂ extract was given orally by gavage to male and female Crl:CD (SD) rats daily for 90 days at concentrations of 0, 30, and 150 mg/kg. None of the groups differed in feed consumption or body weight change. Other than a few variations of some hematological assessments relative to controls, the values were within normal ranges regardless of study group assignment. In the histopathology within the 150 mg/kg group, male animals presented moderate atrophy of reproductive structures. Slight eosinophilic infiltration of kidney tissues was observed in male and female animals at this higher dose. Importantly, this Aloe vera extract was non-mutagenic based on the Ames test, the chromosomal aberration test, and an in vivo bone marrow micronucleus test. Similar in vivo studies have been conducted with an Aloe vera Gel beverage containing 8–10 ppm of Aloin A and B plus other HADs at less than 0.5 ppm with clear negative results for mutagenicity and clastogenicity. In addition, a mixture of purified Aloin A and B was also not genotoxic.

Disclosure statement

The authors are paid scientific consultants to Forever Living Products, which manufacturers products containing Aloe vera. The testing was conducted under their supervision and the manuscript, although reviewed by the company, is the sole work product of the authors.

Additional information

Funding

Aloe Vera, 13745 Jupiter Rd Dallas​, TX, 75238-2418 United States. The authors are paid scientific consultants to Forever Living Products, which manufacturers products containing Aloe vera.